Allogeneic Mesenchymal Stromal Cells Overexpressing Mutant

Transcription

ORIGINAL RESEARCH
Allogeneic Mesenchymal Stromal Cells Overexpressing Mutant
Human Hypoxia-Inducible Factor 1-a (HIF1-a) in an Ovine Model
of Acute Myocardial Infarction
Anna P. Hnatiuk, MD, PhD;* Sang-Ging Ong, PhD;* Fernanda D. Olea, PhD; Paola Locatelli, MD, PhD; Johannes Riegler, PhD;
Won Hee Lee, PhD; Cheng Hao Jen, BSc; Andrea De Lorenzi, MD; Carlos S. Gimenez, MSc; Ruben Laguens, MD, PhD; Joseph C. Wu, MD,
PhD; Alberto Crottogini, MD, PhD
Background-—Bone marrow mesenchymal stromal cells (BMMSCs) are cardioprotective in acute myocardial infarction (AMI)
because of release of paracrine angiogenic and prosurvival factors. Hypoxia-inducible factor 1-a (HIF1-a), rapidly degraded during
normoxia, is stabilized during ischemia and upregulates various cardioprotective genes. We hypothesized that BMMSCs engineered
to overexpress mutant, oxygen-resistant HIF1-a would confer greater cardioprotection than nontransfected BMMSCs in sheep with
AMI.
Downloaded from http://jaha.ahajournals.org/ by guest on November 20, 2016
Methods and Results-—Allogeneic BMMSCs transfected with a minicircle vector encoding mutant HIF1-a (BMMSC-HIF) were
injected in the peri-infarct of sheep (n=6) undergoing coronary occlusion. Over 2 months, infarct volume measured by cardiac
magnetic resonance (CMR) imaging decreased by 71.71.3% (P<0.001), and left ventricular (LV) percent ejection fraction (%EF)
increased near 2-fold (P<0.001) in the presence of markedly decreased end-systolic volume. Sheep receiving nontransfected
BMMSCs (BMMSC; n=6) displayed less infarct size limitation and percent LVEF improvement, whereas in placebo-treated animals
(n=6), neither parameters changed over time. HIF1-a-transfected BMMSCs (BMMSC-HIF) induced angio-/arteriogenesis and
decreased apoptosis by HIF1-mediated overexpression of erythropoietin, inducible nitrous oxide synthase, vascular endothelial
growth factor, and angiopoietin-1. Cell tracking using paramagnetic iron nanoparticles in 12 additional sheep revealed enhanced
long-term retention of BMMSC-HIF.
Conclusions-—Intramyocardial delivery of BMMSC-HIF reduced infarct size and improved LV systolic performance compared to
BMMSC, attributed to increased neovascularization and cardioprotective effects induced by HIF1-mediated overexpression of
paracrine factors and enhanced retention of injected cells. Given the safety of the minicircle vector and the feasibility of BMMSCs
for allogeneic application, this treatment may be potentially useful in the clinic. ( J Am Heart Assoc. 2016;5:e003714 doi:
10.1161/JAHA.116.003714)
Key Words: angiogenesis • growth substances • myocardial infarction
I
schemic heart disease remains the leading cause of death
and disability worldwide.1 Its most severe complication,
acute myocardial infarction (AMI), results in permanent loss of
cardiomyocytes with posterior scar formation, pathological
remodeling, and progression to heart failure.2 Hence, repair
and regeneration of cardiac tissue post-AMI has become a key
objective of gene- and stem cell–based therapies.
To date, bone marrow mesenchymal stromal cells (BMMSCs)
represent the most commonly used cell type in preclinical
research and clinical trials. In addition to their ease of isolation
From the Instituto de Medicina Traslacional, Trasplante y Bioingenierıa (IMETTYB), Universidad Favaloro-CONICET, Buenos Aires, Argentina (A.P.H., F.D.O., P.L., C.S.G.,
A.C.); Departamento de Fisiologıa (A.P.H., F.D.O., P.L., C.S.G., A.C.) and Departmento de Patologıa (R.L.), Facultad de Ciencias Medicas, Universidad Favaloro, Buenos
Aires, Argentina; Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA (S.-G.O., J.R., W.H.L., J.C.W.); St George’s, University of
London, United Kingdom (C.H.J.); Departmento de Cardiologıa, Hospital Universitario de la Fundacion Favaloro, Buenos Aires, Argentina (A.D.L.).
Accompanying Videos S1 through S4 are available at http://jaha.ahajournals.org/content/5/7/e003714.full#sec-35
*Dr Hnatiuk and Dr Ong contributed equally to this work.
Correspondence to: Alberto Crottogini, MD, PhD, Department of Physiology, Favaloro University, Solıs 453, C1078AAI Buenos Aires, Argentina. E-mail:
[email protected]
Received April 18, 2016; accepted May 28, 2016.
ª 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell. This is an open access article under the terms of the Creative
Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is
not used for commercial purposes.
DOI: 10.1161/JAHA.116.003714
Journal of the American Heart Association
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HIF1a Modified MSC in Cardiac Infarction
Hnatiuk et al
Methods
All animal procedures were approved by the Institutional
Animal Care and Use Committee of Favaloro University
(Buenos Aires, Argentina) and performed in accord with the
Guide for Care and Use of Laboratory Animals, 8th edition (US
National Institutes of Health, 2011).
supplemented with 1% (vol/vol) Antibiotic-Antimycotic (Gibco)
and 20% (vol/vol) FBS (Natocor, Cordoba, Argentina) incubated at 37°C in the presence of 5% CO2. After 48 hours,
nonadherent cells were removed, and BMMSCs were subcultured using TrypLE Express (Gibco) until passage 4 and 70% to
80% confluence for subsequent use in all experiments.
Flow Cytometry Characterization of BMMSCs
BMMSCs were labeled with mouse anti-sheep CD44 (AbDSerotec, Raleigh, NC), mouse anti-human CD166 (BD Biosciences, San Diego, CA), and mouse anti-sheep CD45
(AbDSerotec) and characterized by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ). Sample data
were acquired and analyzed using CellQuest-Pro (BD Biosciences) and Cyflogic 1.2.1 software (CyfloLtd, Turku,
Finland).
Adipogenic, Chondrogenic and Osteogenic
Differentiation
BMMSCs were differentiated into adipocytes, chondrocytes,
and osteocytes using the corresponding StemPRO differentiation kits (Gibco), according to the manufacturer’s protocols.
Histological staining was performed with Oil Red for
adipocytes, Alcian Blue for chondrocytes, and Alizarin Red
for osteocytes.
Minicircle Vector and Cell Transfection Efficiency
For cell transfection a novel nonviral MC carrying a mutant
version of human hypoxia inducible factor 1-alpha (mHIF1-a)
was used.11–13 The construct is based on site-specific
mutation of proline 402 and proline 564 (P402A/P564G)
preventing O2-dependent proline hydroxylation and subsequent proteasomal degradation as previously described.13
Lipofectamine LTX (2.5 lL; Invitrogen, Carlsbad, CA) and
Plus Reagent 2 lL for every 2 lg of MC-DNA were used for
transfecting ovine BMMSCs.
Transfection Efficiency
Isolation and Culture of Ovine BMMSCs
Bone marrow aspirates were aseptically harvested from the
iliac crest of donor Corriedale male sheep under general
anesthesia (premedication: intramuscular acepromazine maleate 5 mg; induction: intravenous propofol 3 mg/kg; maintenance: 2% isoflurane in oxygen under mechanical ventilation).
Bone marrow mononuclear cells were obtained by density
gradient centrifugation using Ficoll-Paque Premium (GE
Healthcare, Uppsala, Sweden). Isolated cells were cultured
in low-glucose (1 g/L) DMEM (Gibco, Carlsbad, CA)
DOI: 10.1161/JAHA.116.003714
Transfection efficiency was assessed by transfecting
BMMSCs with either MC encoding green fluorescent protein
(GFP)14 or a conventional plasmid vector carrying GFP (pCruz
GFP; Santa Cruz Biotechnology, Santa Cruz, CA) by flow
cytometry analysis on days 1, 3, and 6 post-transfection.
In Vitro Human Mutated HIF1-a Expression
BMMSCs transfected with mHIF1-a as described above
(BMMSC-HIF) and na€ıve BMMSCs (BMMSC) were cultured in
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and amplification, BMMSCs display immunomodulation capacity,3 multilineage potential including differentiation into cardiomyocytes,4 and ability to release a large number of growth
factors involved in neovasculogenesis and myocardial repair.5
Furthermore, their immune privilege makes them amenable for
allogeneic transplantation.6 Given that BMMSCs are known to
exert their beneficial effect mainly through the release of
paracrine factors, genetic modifications promoting overexpression of cardioprotective growth factors and signaling molecules
may enhance their regenerative potential.7
Under hypoxic conditions, HIF1, a basic-helix-loop-helixPAS heterodimer protein, is a transcriptional activator of a
plethora of downstream genes involved in oxygen homeostasis, angiogenesis, cell proliferation and viability, as well as
tissue remodeling and erythropoiesis.8 Its stability and
transcriptional activity depend on the intracellular oxygen
concentration sensed by the regulatory subunit HIF1-a.
During hypoxia, HIF1-a remains stable, but in well-oxygenated
conditions, von Hippel-Lindau protein binds to HIF1-a, leading
to its ubiquitination and final degradation in the proteasome.9,10
Despite being stable in the post-AMI hypoxic environment,
HIF1-a progressively declines over the next 2 to 3 days and
loses its beneficial effects.10 Hence, we hypothesized that
BMMSCs overexpressing a stable, oxygen-resistant form of
HIF1-a may exhibit greater cardioprotective effects than na€ıve
BMMSCs over a 2-month follow-up period. To facilitate
potential translation to the clinic, we used a novel nonviral
minicircle vector (MC) that displays high transfection efficiency and prolonged transgene expression with the additional advantage of not harboring bacterial sequences
typically associated with conventional plasmids.11
Hnatiuk et al
ORIGINAL RESEARCH
Figure 1. A, Left panel: nontransfected ovine bone marrow mesenchymal stromal cells (BMMSCs) at P4 and 70%
confluence. Right panel: HIF1-a-transfected BMMSCs at P4 and 70% to 80% confluence. Bars: 100 lm. B, Left
panel: ovine antero-apical infarct (yellow dots indicate injections sites). Right panel: injection procedure in periinfarct zone using a 1-mL syringe. HIF indicates hypoxia-inducible factor.
6-well dishes (Figure 1A). Cells were lyzed with buffer RTL.
Total RNA was extracted using RNeasy mini kit (Qiagen,
Hilden, Germany) and reverse-transcription quantitative polymerase chain reaction (RT-qPCR; StepOne Real-Time PCR
System; Applied Biosystems, Foster City, CA) was done to
quantify mRNA levels of HIF1-a at days 1, 3, and 6 posttransfection. Two sets of primers were used to detect HIF1-a
(forward, 50 -CTCAGCCCCAGTGCATTGTA-30 ; reverse, 50 -GAA
CCTCCTATAGCCACCGC-30 ) for wild type and (forward, 50 TTTACCATGCCCCAGATTCAG-30 ; reverse 50 -GGTGAACTTTGTCTAGTGCTTCCA-30 ) for mutant HIF1-a. GAPDH was used as
qPCR amplification endogen control (forward, 50 -GGTCGTC
TCCTGCGACTTCA-30 reverse, 50 -GCCCCAGCATCGAAGGT-30 ).
DOI: 10.1161/JAHA.116.003714
All primers were designed using Primer Express software
(V3.0; Applied Biosystems).
Immunoblotting and In Vitro Hypoxia
BMMSCs were maintained in normoxic conditions or exposed
to hypoxic conditions (BD GasPak EZ System; Becton
Dickinson) for 24 hours as previously described.15 Upon
completion, cells were washed and lysed for immunoblotting,
which was performed with HIF1-a (NB100-105, 1:500; Novus
Biologicals LLC, Littleton, CO) and a-tubulin (9099, 1:1000;
Cell Signaling Technology, Inc., Danvers, MA). Forty micrograms of proteins were loaded. Expression was normalized to
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Hnatiuk et al
Surgical Preparation and Study Protocol
Eighteen male Corriedale sheep, weighting 26.70.7 kg, were
premedicated with intramuscular acepromazine maleate 5 mg.
Anesthesia was induced with intravenous propofol 3 mg/kg
and maintained with 2% isoflurane in oxygen under mechanical
ventilation (Neumovent, C
ordoba, Argentina). A sterile thoracotomy was performed at the fourth intercostal space, the
pericardium was opened, and an anterior-apical infarct was
induced by permanent ligation of branches of the left anterior
descending (LAD) coronary artery, avoiding the first diagonal
branch (Figure 1B). The LAD itself was not ligated to prevent
septal infarction. Thirty minutes after ligation, sheep were
randomized into 3 treatment groups (n=6 per group): PBS
(placebo group), 29107 nontransfected BMMSCs (BMMSC
group), and 29107 HIF1-a-transfected BMMSCs (BMMSC-HIF
group). All treatments, diluted in a final volume of 2 mL of PBS,
were delivered by direct intramyocardial transepicardial injection, divided into 10 aliquots of 200 lL each. Injections were
performed in the normoperfused myocardium surrounding the
ischemic area within 5 mm of the infarct border, which was
readily visible (Figure 1B) and the needle was retained in the
myocardium for 30 seconds after each injection to minimize
leakage. The nature of the injections was kept blind for all
investigators involved in data analysis and processing. Subsequently, the thoracotomy was repaired without pericardial
closure, and, after removal of the tracheal tube, cephalotin 1 g
intravenously was injected and sheep were returned to the
animal house under analgesic treatment (nalbuphine 0.3 mg/
kg, subcutaneous).
Cardiac Magnetic Resonance Procedure
One, 30, and 60 days post-infarction, cardiac magnetic
resonance (CMR) imaging was carried out to assess for
infarct size and left ventricular (LV) function. CMR was
performed with a 1.5 Tesla clinical magnetic resonance
scanner (Achieva; Philips, Amsterdam, The Netherlands). To
analyze cardiac function, two-dimensional cine steady-state–
free precession images with electrocardiogram gating and
manual breath hold were acquired in long- and short-axis
orientations. A total of 12 to 13 short-axis slices spanning the
heart from apex to base and measuring 7 mm in thickness
were acquired without gaps using the following imaging
parameters: field of view (FOV), 25925 cm2; matrix size,
1489120; repetition time (TR), 3.6 ms; echo time (TE),
1.78 ms. To visualize the scar, an intravenous injection of
gadolinium-based contrast (0.2 mmol/kg, Dotarem; Temis
DOI: 10.1161/JAHA.116.003714
Lostalo, Buenos Aires, Argentina) was administered and
delayed contrast-enhanced images were acquired with a
phase-sensitive inversion recovery sequence starting 20 minutes after contrast injection.
The image data for each animal were combined, anonymized, and analyzed with a semiautomated segmentation
method17 using the freely available cardiac analysis software,
Segment (version 1.8 R1172; http://segment.heiberg.se).
Ischemic initial region and infarct size were expressed as
milliliters of infarcted tissue. For cardiac function, LV enddiastolic volume (EDV), end-systolic volume (ESV), and LV
percent ejection fraction (%EF) were measured. Long- and
short-axis videos of each heart at 1, 30, and 60 days
postinjection were recorded.
Short- and Long-Term Cell Tracking
To assess for short-term retention of injected cells, 8 additional
sheep were operated as described above and sacrificed 7 days
postsurgery. BMMSC and BMMSC-HIF were labeled with Cell
Tracker Red CMTPX Dye (Invitrogen) at 0.5-lmol/L concentration in serum-free media according to the manufacturer’s
protocol and injected in the peri-infarct zone (4 animals per
group). Cells were resuspended in PBS after the final washing
step for intramyocardial delivery (29107 cells in 2 mL).
Myocardial samples from the injected sites were harvested
and processed for histological analysis. Given that this shortterm tracking yielded positive results (see Results), we decided
to measure long-term retention. To this aim, another 8
additional sheep, surgically prepared as described above, were
injected in the peri-infarct zones with BMMSC (n=4) or BMMSCHIF (n=4) labeled as follows: after overnight incubation with
0.1 mg/mL of superparamagnetic iron oxide nanoparticles
(SPIO) in low-glucose DMEM (FluidMAG-D; Chemicell, Berlin,
Germany), culture plates were washed 3 times with PBS before
cells were detached for 3 additional washing steps. Cells for the
BMMSC-HIF group were transfected with MC-HIF1-a and
6 hours after transfection labeled with SPIO. After the final
washing step, cells were resuspended at a final concentration of
29107 cells in 2 mL of PBS and stored on ice until intramyocardially injected in the peri-infarct zone.
CMR Tracking of Magnetically Labeled BMMSCs
To visualize SPIO-labeled cells, CMR was performed at 1, 30,
and 60 days postsurgery with the equipment described
above. The T2*-weighted images were acquired using a
multiecho gradient echo sequence with the following imaging
parameters: FOV, 25925 cm2; matrix size, 1409104; TR,
16 ms; and TE, 6.6 ms. The image data for each animal were
combined and anonymized for analysis using a manual regionof-interest segmentation method quantifying the volume (mL)
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ORIGINAL RESEARCH
a-tubulin for whole-cell lysate and densitometry analysis was
performed using ImageJ software (National Institutes of
Health [NIH], Bethesda, MD).16
Hnatiuk et al
Quantification of Myocardial Cell Retention
Cells labeled for 24 hours with FluidMAG-D were washed 3
times, detached, and resuspended in PBS. For T2* measurements, 250 lL of increasing concentration of SPIO-labeled cell
suspension (29106–1.259105) were mixed with 250 lL of 2%
low-melting-point agarose and filled into 1-mL tubes. Sample
tubes were placed into a sample box filled with water to reduce
susceptibility effects. Images were performed using a 1.5T
clinical magnetic resonance scanner described above. For T2*weighted images, we used a multiecho gradient echo sequence
with the same imaging parameters described previously for
CMR tracking of SPIO-labeled BMMSCs in vivo. The number of
labeled cells was quantified by measuring hypointense signal of
each tube using the free available software, ImageJ (NIH), to
estimate the number of cells corresponding to each image’s
intensity. The calibration curve was performed based on the
relation between the number of labeled cells and hypointensity
signal and used to estimate the number of retained labeled cells
into the myocardial tissue corresponding to hypointense signal
emitted in the CMR of each animal at each time point of the
follow-up (Figure 2).
Histology
After completing their respective protocols, sheep were
premedicated with intramuscular acepromazine maleate
(10 mg total) and sacrificed with an overdose of propofol
(13–20 mg/kg) followed by an intravenous bolus injection of
potassium chloride (60 mEq) to arrest the heart in diastole.
Sheep undergoing the main protocol were sacrificed after
the final CMR scanning (60 days post-AMI). The heart was
excised, the atria and the right ventricle were removed, and
the LV was opened along the posterior interventricular sulcus,
extended flat to exhibit its endocardial aspect and photographed. After 24-hour fixation in 4% formaldehyde at room
temperature, 2 myocardial samples measuring 2 cm2 (one
including the fibrotic scar and the peri-infarct zone and the
other only normoperfused tissue remote from the scar) were
embedded in paraffin and cut into 4-lm sections. For
immunohistochemistry, tissue sections were deparaffinized
and brought to PBS solution (pH 7.2). Endogenous peroxidase
was blocked with 3% H2O2 in methanol, antigens were
retrieved with citrate buffer pretreatment in a microwave
oven, and sections were incubated during 1 hour with a
specific monoclonal antibody against smooth-muscle actin
DOI: 10.1161/JAHA.116.003714
(BioGenex, Fremont, CA) to identify arterioles (vessels measuring 10–100 lm in diameter displaying a smooth-muscle
media layer), and the endothelial marker, Biotinylated Euonymous Europaeus Lectin (Vector Laboratories, Burlingame, CA),
to identify capillaries (vessels with only an endothelial wall
measuring up to 10 lm in diameter). Random fields from the
peri-infarct area were examined at 920 magnification for
arterioles and capillaries under light microscopy (Axiophot;
Zeiss, Jena, Gremany) with the aid of a computer-assisted
image-analysis program (Zeiss AxioCam MRc5 camera and
AxioVision software [v4.7.2.0]; Carl Zeiss MicroImaging, Inc.,
Thornwood, NY). The scanned area was 151.623.1 mm2 for
the placebo group, 166.122.7 mm2 for BMMSC, and
173.540.8 mm2 for BMMSC-HIF. Vascular density was
expressed as number of capillaries and arterioles per mm2.
In each experimental group, the cross-sectional diameter
(CSD; lm) and the lumen diameter (LD; lm) in 20 arterioles
per animal were measured at 7 and 60 days. From CSD and
LD, wall thickness (WTh; lm) was calculated according to the
equation: WTh=(CSDLD)/2.
Picrosirius red staining was used to quantify fibrosis in
normoperfused zones remote from injections sites for the
long-term functional protocol hearts. Random fields were
examined at 910. The scanned area was 486.291.8 mm2
for the placebo group, 539.2113.7 mm2 for BMMSC, and
432.8102.7 mm2 for BMMSC-HIF. All quantifications and
images analyses were performed with ImageJ and ImageProPlus software (v6.0; Media Cybernetics, Silver Spring, MD).
Sheep from the long-term cell tracking protocol were
sacrificed after the final CMR scan (60 days post-AMI). All
hearts were processed as outlined above and stained for
macrophages with mouse anti-human CD68, Clone KP1
(Dako, Glostrup, Denmark), whereas the presence of intracellular iron was detected by Prussian blue staining. Microscopical analysis and quantification were done under 940
magnifications through the whole surface of each section, and
the scanned area was 162.524.0 mm2 for BMMSC and
189.538.0 mm2 for BMMSC-HIF. Sheep sacrificed at 7 days
were used for short-term cell tracking and for immunohistochemistry. For short-term cell tracking analysis, hearts were
fixed in 4% formaldehyde, cryoprotected in a 30% sucrose
solution at 4°C until complete tissue penetration, and
embedded in Cryoplast (Biopack, Buenos Aires, Argentina).
BMMSCs labeled with CellTracker Red CMTPX Dye were
detected by fluorescence microscopy in 7-lm-thick cryosections. For immunohistochemistry, hearts were treated as
above for quantification of arteriolar and capillary densities.
The scanned area was 135.731.7 mm2 for the placebo
group, 142.114.8 mm2 for BMMSC, and 151.141.4 mm2
for BMMSC-HIF. In addition, staining with phospho-histone-3
antibody (Ser10; Cell Signaling Technology) and mouse
monoclonal antibody Ki67 antigen (Leica Biosystems,
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ORIGINAL RESEARCH
of the hypointensity emitted by the injected zones using
Segment software. For all experiments involving SPIO-labeled
BMMSCs, 25% labeled cells were mixed with 75% unlabeled
cells because iron internalization was sufficient to generate
enough CMR signal loss on T2*-weighted images.
ORIGINAL RESEARCH
A
Hnatiuk et al
B
C
D
Figure 2. Magnetic resonance T2* images of tubes filled with agar containing an increasing number of
magnetically labeled cells (A) and the calibration curve of T2* emitted intensity by each tube (B). Images of in vitro
ovine bone marrow mesenchymal stromal cells 25% labeled prepared to injection (C) and 100% labeled (D) with
0.1 mg/mL of superparamagnetic iron oxide nanoparticles. Bars: 200 lm.
Newcastle, UK) to detect cardiomyocyte proliferation were
used. It is important to notice that the immunohistochemistry
studies at 7 days post-AMI included 4 sheep receiving
placebo. Microscopical analysis and quantification were
performed using hematoxylin and Mallory staining (without
acid fuchsin to avoid nuclei staining), respectively, under
9100 magnifications on the whole surface of each section,
and only the cells clearly defined as cardiomyocytes on the
basis of their shape and presence of striations were
considered. To detect apoptosis in paraffin sections of tissue
from peri-infarct zones of placebo, BMMSC and BMMSC-HIF
groups, the terminal deoxynucleotidyl transferase dUTP nick
end labeling (TUNEL) assay was performed using the In situ
DOI: 10.1161/JAHA.116.003714
cell death detection kit (Roche, Basel, Switzerland) detecting
rhodamine-positive nuclei by fluorescence microscopy. Nuclei
were double stained with 4’,6-diamidino-2-phenylindole
(DAPI), and the results are expressed as the percentage of
TUNEL-positive nuclei of the total number of nuclei. The
scanned area was 98.025.1 mm2 for the placebo group,
94.410.3 mm2 for BMMSC, and 85.634.9 mm2 for
BMMSC-HIF.
In Vivo Gene and Protein Expression
In all sheep belonging to the short-term protocol, myocardial
tissue samples from the peri-infarct zone and a zone remote
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Hnatiuk et al
Statistical Analysis
Variables were analyzed with 1-way-ANOVA-Tukey for intergroup comparisons or 2-way-ANOVA-Bonferroni for inter- and
intra-group comparisons, using GraphPad Prism software
(version 5.0; GraphPad Software Inc., La Jolla, CA). Statistical
significance was set at P<0.05 (2-tailed). Data are expressed
as meanSEM.
Results
Animal Surgical Outcome
Of a total of 44 operated sheep, 3 died of irreversible
ventricular fibrillation before the intramyocardial injection
procedure, and 3 (1 treated with BMMSC and 2 with placebo)
died of unknown cause during follow-up. These 3 sheep were
replaced with new animals. Consequently, results from 38
sheep are reported. Eighteen sheep were used for the main
protocol, 8 for the long-term cell tracking protocol, and 12 for
the short-term cell tracking and gene expression protocol.
Note that 4 of these 12 sheep are placebo animals that did
not undergo cell tracking.
BMMSCs Were Successfully Characterized and
Transfected
Flow cytometry characterization revealed that the isolated
BMMSCs were positive for CD44, and CD166, but negative for
DOI: 10.1161/JAHA.116.003714
CD45 (Figure 3A). Likewise, BMMSCs were amenable to
differentiation into adipogenic, chondrogenic, and osteogenic
linages after 2 weeks of culture (Figure 3B). Transfection
efficiency measured by GFP fluorescence was higher (P<0.001)
for the minicircle vector compared to conventional plasmids
at day 1 (39.70.7% vs 23.22.4%), day 3 (70.12.4% vs
36.53.2%), and day 6 (79.30.4% vs 25.62.9%) posttransfection (Figure 3C). Moreover, whereas transfection efficiency with MC showed a temporal increase, it decayed
(P<0.05) with the conventional plasmid at day 6 compared to
day 3 (Figure 3C). In BMMSC-HIF, HIF1-a mRNA increased
39106-fold from days 1 to 6, and mRNA expression of wild-type
of HIF1-a was almost undetectable in the BMMSC and BMMSCHIF (Figure 3D). The increase in HIF1-a mRNA expression was
further supported by immunoblotting for HIF1-a protein levels,
which demonstrated a significant increase in BMMSC-HIF
compared to controls (Figure 3E and 3F).
Cell Retention Is Enhanced by HIF1-a-Transfected
BMMSCs
To investigate whether HIF1-a overexpression improves cell
retention, BMMSCs were labeled with fluorescent CMTPX dye
(short-term tracking) or with SPIO detectable as hypointense
zones in T2* CMR sequences (mid- and long-term tracking). At
7 days, BMMSC and BMMSC-HIF were readily visible in injected
areas (Figure 4A). For long-term tracking, CMR in animals
injected with SPIO-labeled BMMSC-HIF and BMMSC were
performed at 1, 30, and 60 days postinjection. CMR showed
retention of injected cells over time. Group analysis showed
that, as expected, the initial volume of hypointense zones was
similar for both groups at day 1 (BMMSC: 1.550.07 mL;
BMMSC-HIF: 1.580.1 mL; P=NS [not significant]), and the
hypointense volume decreased significantly at 30 and 60 days,
compared to day 1 (P<0.001), but BMMSC-HIF presented
significantly higher (P<0.001) T2* hypointense signal than the
BMMSC group at 30 days (1.30.12 vs 0.590.06 mL,
respectively) and at 60 days (1.150.11 vs 0.510.03 mL,
respectively). Estimation of the number of retained cells in
peri-infarct zones showed that BMMSC and BMMSC-HIF had
similar number of cells at day 1 (9 193 326995 774 [45.9%]
vs 9 774 688859 426 [48.8%], respectively; P=NS).
Sheep receiving BMMSC-HIF presented a higher number of
retained cells than BMMSC at day 30 (6 296 250965 348
vs 3 076 365127 537; P<0.001) and day 60 (5 855
1081 126 621 vs 2 808 17989 615; P<0.001), indicating
that 14.04% of injected cells in the BMMSC group and 29.27% in
the BMMSC-HIF group were retained at the end of follow-up
(Figure 4B through 4D).
To evaluate whether the detected iron corresponded to
BMMSCs effectively retained in the myocardium or to iron
particles phagocytized by macrophages, 2 consecutive
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ORIGINAL RESEARCH
to the infarct were taken and stored in liquid nitrogen at
80°C. RNA was extracted using the RNeasy minikit (Qiagen)
and RT-qPCR was done to quantify gene expression of both
mutated and wild-type HIF1-a and the downstream angiogenic
factors erythropoietin (EPO), inducible nitrous oxide synthase
(iNOS), vascular endothelial growth factor (VEGF), and
angiopoietin-1 (ANGP-1), using the appropriate following
primers: EPO (forward, 50 -ACGATGGGCTGTGCAGAAG-30 ;
reverse, 50 -CCTTGGTGTCTGGGACAGTGA-30 ); iNOS (forward,
50 -GATGCAGAAGGCCATGTCATC-30 ; reverse, 50 -CTCCCTGTCT
CTGTTGCAAAGA-30 ); VEGF (forward, 50 -GCCCACTGAGGAGTTC
AACATC-30 ; reverse, 50 -CTGGCTTTGGTGAGGTTTG-30 ); ANGP-1
(forward, 50 -CAGAGCAGACCAGGAAGTT-30 ; reverse, 50 -TCCAG
CAGTTGTATTTCAAGTCG-30 ). The primers for wild-type and
mutated form of HIF1-a and GAPDH have been described
above.
To detect the presence of angiogenic proteins, arrays
spotted with human-specific antibodies (Proteome Profiler
Human Angiogenesis Antibody Array; ARY007; R&D Systems,
Minneapolis, MN) were performed according to the manufacturer’s protocol in peri-infarct myocardial tissue lysates from
each experimental group.
Hnatiuk et al
ORIGINAL RESEARCH
A
CD166+
82.45%
CD44+
94.36 %
CD45+
6,39 %
B
Adipocyte lineage
C
E
Chondrocyte lineage
Osteocyte lineage
D
F
Figure 3. Ovine bone marrow mesenchymal stromal cells (BMMSC) characterization and transfection
efficiency. A, BMMSCs were CD44+, CD166+, and CD45. B, Differentiation of BMMSC into adipocytes
(left), chondrocytes (mid), and osteocytes (right). Bars: 200 lm. C, Transfection efficiency (TE) at 1, 3, and
6 days post-transfection of the minicircle-GFP (MC-GFP) as compared with conventional plasmid-GFP (pGFP). *P<0.001 vs p-GFP; †P<0.01 vs day 1 and day 6; ‡P<0.01 vs day 1 and day 3; §P<0.05 vs day 3. D,
Expression of mutant form (mf) and wild-type form (wt) of HIF1-a (RT-qPCR) at 1, 3, and 6 days posttransfection. *P<0.001 vs BMMSC; †P<0.001 vs day 1 and day 3. E, Representative immunoblot of HIF1-a
in BMMSC, BMMSC-HIF, and BMMSC exposed to hypoxia for 24 hours. F, Densitometry analysis of (E)
(*P<0.05). GFP indicates green fluorescent protein; HIF, hypoxia-inducible factor; RT-qPCR, reversetranscriptase quantitative polymerase chain reaction.
DOI: 10.1161/JAHA.116.003714
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B
ORIGINAL RESEARCH
A
Hnatiuk et al
C
D
E
F
Figure 4. Cell retention. A, Short-term (7 days) tracking: injected cells stained with Red CMTPX dye
are readily visible in nontransfected bone marrow mesenchymal stromal cells (BMMSC; upper) and
HIF1-a-transfected bone marrow mesenchymal stromal cells (BMMSC-HIF; lower) groups (bars:
50 lm). B and C, Long-term cell retention: In the BMMSC-HIF group (n=4), the number of cells and
hypointense volume remains higher than in the BMMSC group (n=4) over time. *P<0.001 vs day 1;
†
P<0.05 vs BMMSC; ‡P<0.001 vs day 1; §P<0.001 vs day 1 and vs BMMSC. D, Long-term tracking:
BMMSC marked with superparamagnetic oxide iron nanoparticles are visible (arrowheads) at 1, 30,
and 60 days postinjection. E, Histological staining with Prussian blue and anti-CD68 antibody. BMMSC
group (n=4) and BMMSC-HIF group (n=4). *P<0.05 vs BMMSC; †P<0.05 vs anti-CD68. F,
Representative Prussian-blue–stained slices (left) and anti-CD68-stained slices (right) of an animal
of BMMSC (upper) and BMMSC-HIF (lower) groups. DAPI indicates 4’,6-diamidino-2-phenylindole; HIF,
hypoxia-inducible factor.
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ORIGINAL RESEARCH
paraffin-embedded tissue sections 4 lm in thickness were
stained; one with Prussian blue and the other with an antibody
against CD68. Whereas the number of CD68-positive cells
was similar for BMMSC (2.020.51 cells/mm2) and BMMSCHIF (1.270.23 cells/mm2; P=NS), Prussian blue–positive
cells were more abundant in the samples from BMMSC-HIF
(5.01.67 cells/mm2 vs 1.080.09 cells/mm2; P<0.05;
Figure 4E), suggesting that, in BMMSC-HIF, a significant part
of the detected iron effectively corresponded to the injected
cells (Figure 4F).
A
BMMSC-HIF Induced the Largest Reduction in
Infarct Size
1 day
30 days
60 days
MSCs
BMMSC
Placebo
B
BMMSC - HIF
Infarct size, calculated from CMR and expressed in milliliters
of infarcted tissue, is depicted in Figure 5A. The initial
ischemic region after 1 day post-AMI was similar in all groups
(placebo: 9.10.2 mL; BMMSC: 9.40.4 mL; BMMSC-HIF:
9.60.3 mL; P=NS). Compared to placebo, infarct size was
smaller by 18.8% for BMMSC and 49.6% for BMMSC-HIF at
30 days post-treatment, and 36.3% and 66.6%, respectively,
at end of follow-up. At both 30 and 60 days, the absolute
values for infarct size in BMMSC and BMMSC-HIF were
significantly lower than placebo. Paired comparisons showed
that the scar-size reduction from the initial ischemic region at
day 1 to 60 days scar size was 44.14.7% for the BMMSC
group and 71.71.3% for BMMSC-HIF. By contrast, the
placebo group did not show significant reductions between
the initial ischemic damage at day 1 and final scar size at day
60 postinjection. Examples of LV short-axis CMR images are
shown in Figure 5B, and examples of LV endocardial aspect of
infarct size are depicted in Figure 5C.
C
Placebo
BMMSC
BMMSC - HIF
BMMSC-HIF Improved Cardiac Function
EDV did not differ among groups at day 1 (placebo:
63.22.6 mL;
BMMSC:
62.13.1 mL;
BMMSC-HIF:
63.061.9 mL; P=NS). The placebo group exhibited increased
EDV at day 30 (70.52.5 mL) and day 60 (74.51.0 mL)
compared to day 1 (P<0.05 and <0.01, respectively). In
BMMSC and BMMSC-HIF groups, EDV did not change over time
(P=NS; Figure 6A). ESV was similar among groups at day 1
(placebo: 46.72.4 mL; BMMSC: 45.22.2 mL; BMMSC-HIF:
45.71.4 mL; P=NS). In the BMMSC group, ESV was lower
than in the placebo group at end of follow-up (BMMSC:
42.91.1; placebo: 52.12.1 mL; P<0.01). In the BMMSC-HIF
group, ESV decreased at day 30 (38.20.9 mL) and further at
day 60 (29.01.7 mL) compared to its baseline value (P<0.01
and <0.001, respectively). In addition, it was lower than
placebo at 30 days (P<0.001) and also lower (P<0.001) than
placebo and BMMSC at 60 days (Figure 6B). As a result of the
relative changes in EDV and ESV, %EF increased from
DOI: 10.1161/JAHA.116.003714
Figure 5. A, Ischemic region and infarct size assessed from
cardiac magnetic resonance (CMR). Extent of scar size decrease
is significantly larger in the group treated with HIF1-a transfected
bone marrow mesenchymal stromal cells (BMMSC-HIF) at 30 and
60 days postinfarction. Placebo group (n=6), BMMSC group
(n=6), and BMMSC-HIF group (n=6). *P<0.001 vs day 1; †P<0.001
vs day 30; ‡P<0.01 vs placebo; §P<0.001 vs placebo; ||P<0.001 vs
placebo and BMMSC groups. B, Representative short-axis LV
images (late gadolinium enhancement) at end diastole at 1, 30,
and 60 days post-treatment. The animal from the BMMSC-HIF
group displays the maximum scar-size reduction. Infarct borders
are dotted in yellow; red line indicates endocardium and green line
indicates epicardium. C, Endocardial aspect of representative
hearts from each group opened along the posterior interventricular sulcus. The pale zone corresponds to the infarct scar. HIF
indicates hypoxia-inducible factor; LV, left ventricle.
10
Hnatiuk et al
BMMSC-HIF Promoted Microvascular
Proliferation
Microvascular densities were assessed in sheep sacrificed at
7 and 60 days post-treatment. Compared to placebo and
BMMSC, arteriolar and capillary densities were significantly
increased in BMMSC-HIF at indicated time points (P<0.001;
Figure 7A and 7B). The arterioles of animals from the
BMMSC-HIF group presented higher CSD and WTh at 7 and
60 days post-treatment (P<0.001) without significant
changes in LD. Both WTh and CSD were higher at day 60
compared to day 7 (P<0.001). In the BMMSC group, the
arterioles exhibited higher WTh at day 7 compared to placebo
(P<0.05), without significant changes at day 60 posttreatment. In the placebo group, arteriolar diameters and
WTh remained stable over time (P=NS). Representative
histological images are shown in Figure 7C, and the absolute
values are listed in Tables 1 and 2. In remote zones, there
were no significant differences for capillary or arteriolar
densities at any time points (data not shown).
BMMSC-HIF Reduced Apoptosis in the Heart and
Induced Cell Proliferation
To measure apoptosis in the peri-infarct, TUNEL assay was
performed at 7 days post-treatment (Figure 8A). Apoptosis
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was 53.374.2% in placebo, 35.321.64% in BMMSC
(P<0.01 versus placebo), and 19.60.37% (P<0.001) in
BMMSC-HIF.
On the other hand, the percent cardiomyocyte nuclei
positive for the mitotic marker, pH3, was highest in
BMMSC-HIF (50.085.13%/mm2), followed by BMMSC
(34.632.69%/mm2) and placebo (15.741.41%/mm2;
P<0.001 vs BMMSC-HIF and P<0.05 vs BMMSC; Figure 8B).
In addition, the presence of Ki67-positive cardiomyocytes
nuclei were also increased in BMMSC-HIF (placebo: 0.22
0.03 cells/mm2; BMMSC: 0.250.07 cells/mm2; BMMSCHIF: 0.50.05 cells/mm2, P<0.05 vs placebo and BMMSC;
Figure 9A and 9B). Some of the Ki67-positive nuclei showed
ongoing mitosis (Figure 9C).
HIF1-a Transfection Enhanced the Expression of
HIF1 Downstream Genes
Mutated HIF1-a mRNA expression was significantly higher in
the peri-infarct area of animals treated with BMMSC-HIF and
was undetectable in the remote normoperfused zones, as well
as in sheep from BMMSC and placebo. mRNA of wild-type
HIF1-a was barely detectable in the peri-infarct zones and in
the remote myocardium of all groups. HIF1 downstream
genes involved in angiogenesis and tissue repair (EPO, iNOS,
VEGF, and ANGP-1) were also significantly upregulated in the
peri-infarct of animals treated with BMMSC-HIF (P<0.01).
Whereas EPO and iNOS were detectable only in BMMSC-HIF,
VEGF and ANGP-1 were also upregulated in BMMSC-treated
animals, albeit at a lower level compared to BMMSC-HIF.
Expression levels of HIF1-a and the aforementioned genes in
peri-infarct and remote zones are shown in Figure 10.
Discussion
Our results showed that intramyocardial delivery of allogeneic
BMMSCs overexpressing a mutant, oxygen-resistant form of
HIF1-a reduced infarct size and improved LV function to a
larger extent than BMMSCs in sheep with AMI at 2 months
post-treatment, which was associated with increased neovascularization and reduced apoptosis in the heart along with
increased expression of proangiogenic genes. In animal
models of AMI, intramyocardial implant of unmodified
BMMSCs has been shown to induce variable infarct-size
reductions. In some studies, reductions of over 50% have
been reported.18,19 In our BMMSC-treated animals, at
2 months post-treatment, infarcts were 36% smaller than in
sheep receiving placebo, and 44% smaller with regard to their
initial size. However, the extent of scar-size limitation was
significantly larger for animals receiving BMMSC-HIF (66%
compared to placebo at 60 days and 71% compared to initial
postinfarct size). Given that the infarct-limiting effect
11
ORIGINAL RESEARCH
27.11.3% at day 1 to 36.71.1% at day 30 and to 37.81.0%
at day 60 (both P<0.001) in the BMMSC group, and from
27.22.6% at day 1 to 42.40.9% at day 30 and further to
53.32.0% at day 60 (both P<0.001) in the BMMSC-HIF group.
Both groups displayed significant differences compared to
placebo (P<0.05 and <0.01, respectively) at both time points.
However, at the end of follow-up, %EF was highest in BMMSCHIF and significantly different (P<0.001) not only from placebo,
but also from BMMSC (Figure 6C). Video recordings of CMR
scans at 1 and 60 days post-treatment showed that BMMSCHIF induced a noticeable recovery of wall motion in the
infarcted zone, whereas in the placebo-treated animals, the
infarcted wall remained akinetic. Selected illustrative images
taken from videos of an animal of each group are shown in
Figure 6D. Additionally, CMR movies of a placebo-treated
(Videos S1 and S2) and a BMMSC-HIF-treated animal (Videos
S3 and S4) are provided.
As a measurement of interstitial fibrosis in normoperfused
zones remote to AMI, Picrosirius red–stained areas were
significantly higher (P<0.001) in the placebo group
(8.460.62%) than BMMSC (4.711.16%) and BMMSC-HIF
(2.780.27%; P<0.001 vs BMMSC), indicating that the
placebo group underwent significant remodeling over
2 months postinfarction, which was prevented by both
BMMSC and BMMSC-HIF treatments (Figure 6E and 6F).
Hnatiuk et al
B
C
D
ORIGINAL RESEARCH
A
E
F
Figure 6. Left ventricular function. A, End diastolic volume (EDV); *P<0.05 vs day 1; †P<0.01 vs day 1;
‡
P<0.001 vs placebo. B, End systolic volume (ESV): *P<0.01 vs day 1; †P<0.001 vs day 1; ‡P<0.01 vs day 30;
P<0.001 vs placebo; ||P<0.01 vs placebo; #P<0.001 vs placebo and BMMSC. C, Ejection fraction (%EF):
*P<0.001 vs day 1; †P<0.001 vs day 30; ‡P<0.05 vs placebo; §P<0.001 vs placebo; ||P<0.001 vs placebo;
#
P<0.001 vs placebo and BMMSC. D, Left ventricular function as assessed by cardiac magnetic resonance
(CMR). Representative short-axis images at 1 and 60 days postinfarction taken from video recordings by a
placebo, a BMMSC, and BMMSC-HIF animal. At day 1, the akinetic zone (arrowheads) was similar among groups.
At day 60, the zone showing akinesia on day 1 displayed systolic thickening in the BMMSC-HIF-treated animal.
Note that precise infarct limits are not seen because video recordings are taken before late gadolinium
enhancement. For complete short- and long-axis video recordings, please see Videos S1 through S4. BMMSC:
group receiving nontransfected bone marrow mesenchymal stromal cells; BMMSC-HIF: group receiving HIF1-atransfected bone marrow mesenchymal stromal cells. E, Interstitial fibrosis. Fibrosis area (%) is higher in placebo
group than BMMSC and BMMSC-HIF at 60 days post-treatment (*P<0.01 vs placebo; †P<0.01 vs BMMSC;
‡
P<0.001 vs placebo). F, Placebo group (n=6), BMMSC group (n=6), and BMMSC-HIF group (n=6). ED indicates
end diastolic; ES, end systolic; HIF, hypoxia-inducible factor.
§
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ORIGINAL RESEARCH
A
B
Placebo
BMMSC
BMMSC-HIF
60 days
Capillaries
7 days
60 days
Arterioles
7 days
C
Figure 7. Microvascular density. A, Arteriolar density is highest in BMMSC and BMMSC-HIF at 7 and
60 days post-treatment (*P<0.05 vs placebo; †P<0.001 vs placebo and BMMSC; ‡P<0.01 vs placebo;
P<0.05 vs day 7; ||P<0.001 vs placebo and vs day 7; #P<0.01 vs BMMSC). B, Capillary density is higher at 7
and 60 days in BMMSC and BMMSC-HIF (*P<0.01 vs BMMSCs; †P<0.001 vs placebo; ‡P<0.01 vs placebo;
§
P<0.001 vs placebo and BMMSC). C, Representative histological images from an animal of each group at 7
and 60 days showing arterioles (smooth-muscle actin antibody/hematoxilin; bars: 100 lm) and capillaries
(Biotinylated Euonymous Europaeus Lectin antibody/hematoxilin; bars: 20 lm). BMMSC: group receiving
nontransfected bone marrow mesenchymal stromal cells; BMMSC-HIF: group receiving HIF1-a-transfected
bone marrow mesenchymal stromal cells. Note higher arteriolar wall thickness in the BMMSC-HIF group at
60 days. Microvacular density at 7 days: placebo group (n=4), BMMSC group (n=4), and BMMSC-HIF group
(n=4). Microvascular density at 60 days: placebo group (n=6), BMMSC group (n=6), and BMMSC-HIF group
(n=6). HIF indicates hypoxia-inducible factor.
§
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Hnatiuk et al
Arterioles/mm2
Placebo
BMMSC
BMMSCHIF
Capillaries/mm2
Day 7
Day 60
9.10.8
13.41.8
15.01.4*
27.81.5
†
33.63.8
‡§
63.26.9
||#
Day 7
Day 60
1521129
120059
174364‡
198439
2905210*
†
2967129§
Day 7: n=4/group; day 60: n=6/group. BMMSC indicates group treated with
nontransfected bone marrow mesenchymal stromal cells; BMMSC-HIF, group treated
with HIF1-a-transfected bone marrow mesenchymal stromal cells. HIF, hypoxia-inducible
factor.
Arteriolar density: *P<0.05 vs placebo; †P<0.001 vs placebo and BMMSC; ‡P<0.01 vs
placebo; §P<0.05 vs day 7; ||P<0.001 vs placebo and vs day 7; #P<0.01 vs BMMSC.
Capillary density: *P<0.01 vs BMMSC; †P<0.001 vs placebo; ‡P<0.01 vs placebo;
§
P<0.001 vs placebo and BMMSC.
progressed over time, it is plausible to assume that enhanced
long-term cell retention permitted a persistent effect of
overexpressed factors on neovascularization and reduction of
apoptosis in the whole heart.
Importantly, CMR allowed comparisons to be performed on
a paired basis. This was particularly advantageous, because
sheep as a species display heterogeneous LAD artery
distribution, with interindividual variations that make it
difficult to achieve similar infarct sizes across the experimental population.20 The use of CMR at 3 time points allowed us
not only to compare infarct size against the initial ischemic
damage extension, but also to observe how it evolved over
time.
Given that there were no changes in scar size, which was
accompanied with increased EDV over time in placebo group,
LV remodeling occurred in placebo-treated animals. This
remodeling was prevented by injection of both BMMSC and
BMMSC-HIF. Treatment with BMMSC improved LV function at
1 month and prevented EF from deteriorating later on.
However, the largest improvement in LV function was
achieved in BMMSC-HIF. Given that this improvement arose
from a significant 37% decrease in ESV with unchanged EDV,
Table 2. Cross-Sectional Diameter, Wall Thickeness, and Lumen Diameter of Arterioles at 7 and 60 Days Post-Treatment in PeriInfarct Zone
Day 7
Day 60
Placebo
BMMSC
BMMSC-HIF
Placebo
BMMSC
BMMSC-HIF
CSD, lm
17.080.69
21.00.97
27.821.54*
18.091.77
24.061.6
46.112.27*†
LD, lm
6.570.47
6.590.45
6.980.4
7.760.35
7.960.38
7.960.47
5.170.88
8.050.82
19.071.26†‡
WTh, lm
5.250.35
7.20.28*
10.420.75
†
Day 7: n=4/group; day 60: n=6/group. BMMSC: group treated with nontransfected bone marrow mesenchymal stromal cells; BMMSC-HIF: group treated with HIF1-a-transfected bone
marrow mesenchymal stromal cells. HIF indicates hypoxia-inducible factor.
Cross-sectional diameter (CSD): *P<0.001 vs placebo and BMMSC; †P<0.001 vs 7 days. Lumen diameter (LD): no significant difference. Wall thickeness (WTh): *P<0.05 vs placebo;
†
P<0.001 vs placebo and BMMSC; ‡P<0.001 vs 7 days.
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it is reasonable to assume that the angiogenic and improved
cardiac cell proliferation effects of BMMSC-HIF resulted in
increased LV contractile performance. Indeed, an evident
recovery of wall motion and systolic thickening of the
ischemic LV wall at the end of follow-up was observed in
video recordings of CMR scans of these animals (see Videos).
In terms of neovascularization, both BMMSC and BMMSCHIF increased arteriolar density at 7 and 60 days posttreatment, with a significantly larger degree in the latter.
Given that BMMSCs secrete a number of paracrine molecules
involved in migration and proliferation of vascular smoothmuscle cells, enhanced arteriolar growth in animals receiving
BMMSC was expected.21–24 The fact that this effect was
stronger in BMMSC-HIF treated sheep can be attributed to
HIF-mediated overexpression of angiogenic genes as well as
enhanced cell retention, as indicated by our long-term
tracking experiments. Capillary density was also maximal in
BMMSC-HIF at both time points. Interestingly, whereas
arterioles continued to proliferate between day 7 and day
60 in both BMMSC and BMMSC-HIF, capillaries did not
proliferate similarly, and their density remained almost
unchanged with regard to its value at 7 days. This may be
attributed to the fact that ANGP-1, principally involved in
arteriogenesis, displayed larger overexpression than VEGF, a
classic mitogen of endothelial cells. In our study, overexpression of ANGP-1 may have contributed to the arteriolar wall
hypertrophy in the BMMSC-HIF group at the end of follow-up.
Of note, the increase of pH3-positive cardiomyocyte nuclei
observed in the peri-infarct zone in BMMSC-HIF in sheep
sacrificed at 7 days post-treatment may suggest that HIF1-a
downstream genes induce reentry of adult cardiomyocytes
into mitosis, which may contribute to the cardioprotective
effects of BMMSCs. This observation is consistent with
previous studies in rats with AMI reporting HIF1-a-induced
reentry of adult cardiomyocytes into the cell cycle using
mesenchymal stem cells (MSCs) previously transduced with
adenovirus-HIF1-a25 or after direct transduction with adenovirus-HIF1-a.26 In a recent study, a highly significant
correlation was demonstrated between the proliferative
Table 1. Arteriolar and Capillary Density at 7 and 60 Days
Post-Treatment in Peri-Infarct Zone
Hnatiuk et al
ORIGINAL RESEARCH
Figure 8. Heart apoptosis and mitotic cardiomyocytes. A, TUNEL assay. Examples of TUNEL-stained cardiac
tissue from placebo (n=4), BMMSC (n=4), and BMMSC-HIF (n=4) groups. Percentage of apoptotic nuclei (red)
were determined in relation of total number of DAPI-stained nuclei (light blue). Bars: 50 lm. Overall results of
this assay are shown in (C): *P<0.01 vs placebo; †P<0.001 vs placebo and BMMSC. B, Representative images
of phospho-histone-3–positive (pH3+) nuclei (light brown colored nuclei) in peri-AMI tissue from BMMSC-HIF
(n=4), BMMSC (n=4), and placebo (n=4) animals. Bars: 50 lm. Results are shown in (D): *P<0.05 vs placebo;
†
P<0.001 vs placebo and BMMSC. BMMSC: group receiving nontransfected bone marrow mesenchymal
stromal cells; BMMSC-HIF: group receiving HIF1-a-transfected bone marrow mesenchymal stromal cells. AMI
indicates acute myocardial infarction; DAPI, 4’,6-diamidino-2-phenylindole; HIF, hypoxia-inducible factor;
TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.
cardiomyocytes and overexpression of HIF1-a downstream
genes in the peri-infarct myocardium in primates.27 VEGF, one
of the HIF1 downstream genes, has been shown to induce
adult cardiomyocyte mitosis in large mammalian models of
ischemic heart disease.28–30 It is therefore possible that
overexpression of VEGF in our BMMSC-HIF-treated sheep
promoted cardiomyocyte proliferation.
In stem cell–based cardiac regeneration, myocardial cell
retention remains a challenge.31 Among other factors, the cell
delivery technique plays a key role. We treated our sheep
using direct intramyocardial injections, a route shown to yield
significantly higher cell retention than intracoronary and
retrograde coronary venous routes.32,33 However, even when
using the intramyocardial route, it has been reported that
within a few hours, cell retention dropped below 12%.32 We
also observed that VEGF-transfected BMMSCs injected into
the peri-infarct of ovine myocardium, while abundant at
DOI: 10.1161/JAHA.116.003714
7 days, were very scarce 1 month after delivery.24 In this
study, we demonstrate a higher percentage of retained
postinjection cells at day 1, which may be explained by the
implementation of injection technique and also by the
immunomodulatory characteristic of the BMMSCs, which
may reduce the immune response preventing cell death.
In the present study, short-term tracking showed that
BMMSC-HIF and BMMSC were readily visible, confirming our
previous results. Regarding long-term retention, in agreement
with past results,34 we found that it decayed significantly at
days 30 and 60, compared to day 1, in hearts receiving
BMMSC and in BMMSC-HIF, but the percentage of retained
BMMSC-HIF remained higher than BMMSC, which could be
explained by overexpression of prosurvival genes, such as
ANGP-1 and EPO. Prussian blue staining confirmed that ironbearing cells were more abundant in BMMSC-HIF-treated
hearts. To assess whether the enhanced CMR signals arose
15
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ORIGINAL RESEARCH
Figure 8. Continued.
from cardiac macrophages that engulfed the SPIO nanoparticles,35 we quantified CD68-positive cells and found that in
samples of BMMSC-HIF animals, Prussian blue–positive cells
exceeded the amount of macrophages by more than 3-fold.
This was not the case for sheep treated with BMMSC, which
showed no significant differences between Prussian blue–
positive and CD68-positive BMMSCs. Although not conclusive, these observations suggest that a large proportion of the
hypointense CMR signal arose from BMMSC-HIF retained in
the injected myocardium. Our results are in agreement with
those from Drey et al.,36 showing that in mice hearts injected
with SPIO-BMMSCs labeled with a vital stain, the hypointense
zones observed over 4 weeks corresponded to colocalization
of both stains, confirming the retention of SPIO within vital
BMMSCs.36 The enhanced cell retention observed in our
BMMSC-HIF-treated sheep may be attributed to HIF-mediated
overexpression of cell prosurvival genes.25,37 In fact, iNOS, a
key cardioprotective gene in acute and chronic heart
disease,38 displayed a several-fold increased expression. In
rats with AMI, Azarnoush et al. showed that survival and
engraftment of intramyocardially delivered skeletal myoblasts
were dramatically increased when myoblasts were coinjected
with HIF1-a.39 Moreover, it has been recently shown that
overexpression of HIF1-a in human BMMSCs protects against
cell death and apoptosis triggered by hypoxic and oxidative
stress conditions.40
DOI: 10.1161/JAHA.116.003714
Because we did not perform a dose-response curve, we
cannot state whether a higher amount of injected cells would
have resulted in larger cardioprotection. In ovine AMI,
Hamamoto et al. injected 25, 75, 225, and 4509106 MSCs
in the peri-infarct and observed that the lower doses yielded
the largest beneficial effects on infarct size and LV function.41
The amount of cells we injected (209106) was close to the
lowest dose used by Hamamoto et al., and still effective, in
that even without HIF1-a transfection, significant cardioprotection was achieved. Furthermore, in a recent study of
intracoronary MSCs in sheep with AMI, Houtgraaf et al.
showed that at 2 months post-treatment, similar effects on
infarct size, microvascular growth, cardiomyocyte proliferation, and LV systolic and diastolic function were achieved with
doses lower (12.59106), comparable (259106), and higher
(509106) than ours.42 Taken together, these data suggest
that larger amounts of injected cells would not have changed
our results significantly.
Study Limitations
Although the sheep model is increasingly used in cardiovascular translational research, commercially available antibodies against sheep antigens are scarce, which limits precise
characterization of diverse cells. We confined our cellsurface–based identification of BMMSCs to 3 antibodies.
16
ORIGINAL RESEARCH
B
BMMSC
Placebo
A
Hnatiuk et al
BMMSC-HIF
C
Figure 9. Anti-Ki67 staining of peri-infarct zones. A, Ki67-positive cardiomyocyte nuclei (stained brown) in
Mallory-stained slices of the peri-infarct zone of a heart treated with placebo (upper), BMMSC (mid), and
BMMSC-HIF (lower). Bars: 50 lm. B, Ki67-positive cardiomyocytes in treatment groups. *P<0.05 vs placebo and
BMMSC. C, Images of a heart treated with BMMSC-HIF at 940 magnification (bars: 50 lm). Arrows indicate
mitotic figures in adult cardiomyocytes nuclei, which are reproduced in the insets at 9100 magnification (bars:
20 lm). BMMSC: nontransfected bone marrow mesenchymal stromal cells. BMMSC-HIF: transfected BMMSC.
HIF indicates hypoxia-inducible factor.
Nonetheless, further analysis based on their capacity to
differentiate into adipocytes, chondrocytes, and osteocytes
confirmed the mesenchymal nature of the cells. Likewise,
the lack of ovine-specific antibodies prevented thorough
characterization of secreted proteins that may be responsible for the cardioprotective effects observed in this study.
As an alternative, we postulated that anti-human antibodies
may be used to identify some of the secreted proteins.
However, using a protein array spotted with human-specific,
angiogenesis-related antibodies (ARY007; R&D Systems),
only endothelin-1 and fibroblast growth factor were detectable (data not shown). As a consequence, this study is
partially limited by a lack of thorough mechanistic insights in
fully explaining the cardioprotective effects of modified
MSCs, although we suspect increased neovascularization
and reduced apoptosis as a consequence of released
paracrine factors despite limited cell engraftment43 to be
important mediators.
Another limitation of the model is that it does not
reproduce the risk factors and comorbidities usually
DOI: 10.1161/JAHA.116.003714
associated with human ischemic heart disease. The findings
that these features jeopardize the angiogenic efficiency of
bone marrow cells44 may help explain why clinical trials have
failed to reproduce the therapeutic results obtained in animal
models. Finally, a problem associated with the use of the
intramyocardial route, namely, that part of the injectate is
spilled back by systolic contraction, could be attenuated, but
not prevented, by maintaining the needle in place for
30 seconds postinjection.
In summary, intramyocardial injection of allogeneic
BMMSCs overexpressing a mutant, oxygen-resistant form of
HIF1-a reduces infarct size and improves LV systolic performance to a significantly larger extent than BMMSCs in an
ovine model of AMI. These effects are attributed to increased
arteriolar proliferation and attenuated apoptosis induced by
HIF-mediated overexpression of paracrine growth factors that
also enhance long-term myocardial retention of injected cells.
Given the excellent safety profile of the minicircle vector and
feasibility of BMMSCs for allogeneic application, this treatment may be potentially useful in the clinic.
17
Hnatiuk et al
B
C
D
E
F
ORIGINAL RESEARCH
A
Figure 10. Gene expression at 7 days post-treatment. Expression of mutant form (mf) and wild-type form (wt)
of HIF1-a (A), erythropoietin (EPO) (B), inducible nitrous oxide synthase (iNOS) (C), vascular endothelial growth
factor (VEGF) (D), and angiopoietin-1 (ANGP-1) (E) genes in the peri-infarct and remote myocardium, indicating
that, in all cases, gene expression is significantly higher in the peri-infarct zone of the BMMSC-HIF group.
*P<0.01 vs BMMSC and placebo. F, In the peri-infarct, ANGP-1 expression is higher than the other HIF1
downstream genes. *P<0.001 vs EPO, iNOS, and VEGF. A, n=6 per group. B through F, n=4 per group. HIF
indicates hypoxia-inducible factor.
Acknowledgments
Sources of Funding
We thank veterinarians Marıa Ines Besanson and Pedro Iguain for
anesthetic management and animal house assistants Juan Carlos
Mansilla, Osvaldo Sosa, and Juan Ocampo for dedicated care of the
animals. We also thank Julio Martınez, Fabian Gauna, Rosana
Valverdi, and Leonardo Trejo for technical help.
This work was supported by grants PICT-PROBITEC 20112712 and PICT 2011-1181 from the National Agency for the
Promotion of Science and Technology (ANPCyT), Ministry of
Science, Technology and Innovative Production (MINCyT) of
DOI: 10.1161/JAHA.116.003714
18
Hnatiuk et al
Disclosures
angiogenesis, gap junction formation and therapeutic efficacy for myocardial
infarction. Int J Cardiol. 2015;188:22–32.
20. Locatelli P, Olea FD, Mendiz O, Salmo F, Fazzi L, Hnatiuk A, Laguens R,
Crottogini A. An ovine model of postinfarction dilated cardiomyopathy in
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21. Kinnaird T, Stabile E, Burnett MS, Lee CW, Barr S, Fuchs S, Epstein SE.
Marrow-derived stromal cells express genes encoding a broad spectrum of
arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through
paracrine mechanisms. Circ Res. 2004;94:678–685.
None.
22. Ranganath SH, Levy O, Inamdar MS, Karp JM. Harnessing the mesenchymal
stem cell secretome for the treatment of cardiovascular disease. Cell Stem
Cell. 2012;10:244–258.
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Argentina, the Rene Bar
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grateful for the funding support by American Heart Association Postdoctoral Fellowship 15POST22940013 (Ong).
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Allogeneic Mesenchymal Stromal Cells Overexpressing Mutant Human Hypoxia−Inducible
Factor 1−α (HIF1−α) in an Ovine Model of Acute Myocardial Infarction
Anna P. Hnatiuk, Sang-Ging Ong, Fernanda D. Olea, Paola Locatelli, Johannes Riegler, Won Hee
Lee, Cheng Hao Jen, Andrea De Lorenzi, Carlos S. Giménez, Rubén Laguens, Joseph C. Wu and
Alberto Crottogini
J Am Heart Assoc. 2016;5:e003714; originally published July 6, 2016;
doi: 10.1161/JAHA.116.003714
The Journal of the American Heart Association is published by the American Heart Association, 7272 Greenville Avenue,
Dallas, TX 75231
Online ISSN: 2047-9980
The online version of this article, along with updated information and services, is located on the
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Allogeneic Mesenchymal Stromal Cells Overexpressing Mutant

Transcription

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